Fuhrer J P, DeBiasi F, Cooper H L, Schlom J
Biochim Biophys Acta. 1986 May 5;866(4):204-15. doi: 10.1016/0167-4781(86)90045-x.
Protein products of the ras family of oncogenes were immunoprecipitated by an anti-p21 monoclonal antibody, separated by two-dimensional gel electrophoresis and subsequently detected by western immunoblot analysis using the same anti-p21 monoclonal antibody as a probe. Using this method, a 21 kDa oncogene protein (p21) was detected and characterized in cell lines containing Harvey (Ha), Kirsten (Ki), neuroblastoma (N), or cellular (proto) ras genes. The ras gene products from all cell types occurred with multiple forms differing in size, charge or in both parameters. Transforming ras oncogene proteins occurred in easily identifiable groups that were different from each other in molecular weight and charge, were distinctive for each ras gene type and were different from cellular ras equivalents. Similar, but not identical, family groups occurred in different cell types containing the same oncogene. The reproducible occurrence of unpredicted p21 forms suggests that previously unreported post-translational processing steps may be associated with the synthesis of certain oncogene products. This immunoprecipitation/two-dimensional gel/western blot technique is a simple method for the identification and characterization of p21 gene products.
癌基因ras家族的蛋白质产物用抗p21单克隆抗体进行免疫沉淀，通过双向凝胶电泳分离，随后用相同的抗p21单克隆抗体作为探针进行蛋白质印迹分析检测。使用该方法，在含有哈维（Ha）、克里斯滕（Ki）、神经母细胞瘤（N）或细胞（原）ras基因的细胞系中检测并鉴定了一种21 kDa的癌基因蛋白（p21）。所有细胞类型的ras基因产物均以多种形式出现，这些形式在大小、电荷或两个参数上均有所不同。转化型ras癌基因蛋白以易于识别的组出现，这些组在分子量和电荷上彼此不同，对每种ras基因类型具有独特性，并且与细胞ras等同物不同。在含有相同癌基因的不同细胞类型中出现了相似但不完全相同的家族组。未预测到的p21形式的可重复性出现表明，以前未报道的翻译后加工步骤可能与某些癌基因产物的合成有关。这种免疫沉淀/双向凝胶/蛋白质印迹技术是鉴定和表征p21基因产物的一种简单方法。
